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The cyst nematode, Globodera tabacum tabacum Behrens, and the parasitic angiosperm, Egyptian broomrape, Orobanche aegyptiaca Pers., are obligate root parasites that cause severe yield and quality loss of many important crop hosts. Although these represent two diverse classes of parasites, they have significant similarities in the modes of parasitism and complex interactions with their hosts. Conventional control methods have had limited success in controlling these parasites. The overall objective of this research was to engineer resistance to the cyst nematode and Egyptian broomrape by expressing genes encoding parasite specific toxins under the control of parasite-responsive promoters using tobacco (Nicotiana tabacum L. cv. Xanthi). For nematode resistance, an anti-feeding strategy was employed utilizing the tomato proteinase inhibitor I (PI-I) gene as a nematode specific toxin. Transgenic tobacco plants were generated that expressed genes encoding an intracellarly retained or secreted form of tomato PI-I under the control of the nematode-inducible promoter, derived from tomato (Lycopersicon esculentum L.) Hmg2 gene. Our goals were to determine the effectiveness of local PI-I expression on nematode resistance and to determine if intracellular or extracellular PI-I deposition enhances resistance. Two constructs were generated that contained either the coding region of the tomato PI-I gene, lacking the signal sequence (EM1), or the coding region of PI-I including the signal sequence (EM2), fused to the nematode-responsive Hmg2 promoter. Transgenic PI-I plants were inoculated with G. t. tabacum cysts and evaluated for nematode interactions. Our results suggest that local expression of intercellular of PI-I significantly reduced cyst production when compared to the nontransformed controls. For broomrape resistance, a well characterized R/avr gene pair, the tobacco N resistance gene and the tobacco mosaic virus replicase (TMV) gene, was utilized to create novel gene-for-gene resistance via a N gene-mediated hypersensitive response (HR) to limit broomrape parasitism. The bean (Phaselous vulgaris L.) chalcone synthase 8 (CHS8) promoter has been characterized as a broomrape–responsive promoter. We introduced the CHS8:TMV replicase gene construct into tobacco plants that contains an endogenous N gene. Transgenic tobacco plants were inoculated with O. aegyptiaca seeds and monitored for parasite attachment and development. The expression of the TMV replicase leads to a significant reduction in broomrape parasitism. These genetic engineering strategies show promise in enhancing resistance to these destructive parasites. / Ph. D.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are economically important pests of potato (Solanum tuberosum) crops in potato growing regions worldwide. Integrated management is under threat, with effective nematicides increasingly being withdrawn on environmental and health grounds. Alternative strategies are urgently needed and trap cropping could be one of them. The non-tuber-bearing Solanum sisymbriifolium is regarded as an effective trap crop for PCN with strong hatching ability and immunity to PCN infection and has been used in the UK and The Netherlands. However, its mode of action is unknown. In order to shed light on the mode of action so that a novel control strategy could be identified, the interactions between G. pallida and S. sisymbriifolium were investigated using in vitro bioassays. In choice assays, G. pallida J2s were equally attracted to the roots of S. sisymbriifolium and to those of S. tuberosum. However, potato root diffusate (PRD), which is routinely used to induce PCN hatch, failed to attract G. pallida J2s in chemotaxis bioassays, indicating hatching factors (HFs) and soluble compounds present in PRD are not involved in attraction of G. pallida J2s to potato roots. The J2s invaded the roots of S. sisymbriifolium in large numbers but failed to develop further. To facilitate continuous observation of nematode development, a novel in vitro method was devised with the use of Pluronic F-127, which requires no sterilisation, and the life cycle of G. pallida was successfully observed in S. tuberosum roots. Quantitative real-time polymerase chain reaction analyses of defence related genes of S. tuberosum and S. sisymbriifolium infected with G. pallida revealed up-regulation of the chitinase gene (ChtC 2.1) at 3 days post inoculation in S. sisymbriifolium but not in S. tuberosum. Electrospray ionisation-mass spectrometry analyses of root exudate extracts of the two Solanum species and subsequent bioassay-guided fractionation showed that the HF of S. sisymbriifolium differs from that of S. tuberosum. Previously, attention had been solely paid to the hatching ability of the root exudate of S. sisymbriifolium, but this study revealed for the first time that the aerial part extract possesses a significant hatching ability.

2007-2009 metais buvo tiriamas auksinio bulvinio nematodo ir blyškiojo bulvinio nematodo paplitimas Vilniaus regione. Tyrimai buvo atliekami Vilniaus regiono bulvių augintojų, dekoratyvinių, sodo ir miško augalų augintojų ir šiltnamių kontroliuojamuose ūkiuose. Iš vieno hektaro buvo imami 4 dirvožemio pavyzdžiai, o iš bulvių sėklininkystės ūkių ir ūkių, kuriuose auginama dauginimui skirta sodinamoji medžiaga, buvo imami 8 pavyzdžiai. Valstybinės augalų apsaugos tarnybos Fitosanitarinių tyrimų laboratorijoje pavyzdžiai buvo ištirti naudojant centrifugavimo Schuiling‘o centrifugos metodą. Apibendrinus 2007-2009 metų tyrimų duomenis nustatyta, kad buvo ištirtas dirvožemis 428-iuose kontroliuojamuose ūkiuose, kurių bendras tirtų laukų plotas buvo 877,51 ha. Iš viso buvo paimta 4343 pavyzdžiai. Iš kurių 652 buvo užkrėsti auksiniu bulviniu nematodu. Globodera pallida nebuvo identifikuota. Daugiausiai užkrėstų pavyzdžių nustatyta Švenčionių ir Trakų rajonuose (37,5 ir 33,6 % nuo tirtų tame rajone pavyzdžių). Mažiausiai užkrėstas Ukmergės ir Širvintų rajonų (12,03 ir 11,69 % nuo tirtų tame rajone pavyzdžių) dirvožemis. / The research of the spread of Globodera rostochiensis (Wollenweber) Behrens and Globodera pallida (Stone) Behrens was conducted in the Vilnius region in 2007 - 2009. It was done by potato cultivators of the Vilnius region, by cultivators of decorative, garden and forest plants as well as by controlled greenhouse farms. During the research 4 samples were taken from each 1 hectare and 8 samples from potato seed farms as well as farms where planting material was cultivated for the reproduction purposes. Each sample was tested in the Phytosanitary Research Laboratory of the State Plant Protection Service by using the Schuiling centrifuge method. When summarising the research data of 2007 - 2009 it was found out that the soil test was conducted in 428 controlled farms with the total tested field area of 877.51 ha. 4343 samples were taken in total, 652 of which were contaminated with the Golden Nematode. Pale cyst Nematode was not identifyed. The highest number of contaminated samples was found in Švenčionių and Trakų districts (37.5 % and 33.6 % of the tested samples respectively). Meanwhile the least contaminated soil was in Ukmergės and Širvintų districts (12.03% and 11.69% of the tested samples respectively).

Orientador: Sandra Helena Unêda-Trevisoli / Coorientador: Ana Cristina Pinto Juhász / Coorientador: Luciany Favoreto / Banca: João Antônio da Costa Andrade / Banca: Ivana Marino Barbaro / Banca: Jaime Maia dos Santos / Banca: Pedro luiz Martins Soares / Resumo: A soja [Glycine max (L.) Merrill] é uma leguminosa de ciclo anual que apresenta distintas finalidades, dentre elas, uma crescente vertente relaciona-se ao uso na alimentação humana. Há demanda de cultivares de soja com esta destinação com resistência a patógenos e pragas. Em especial destaca-se o nematoide de cisto da soja (Heterodera glycines) que causa expressiva perda na produtividade. Em virtude do exposto, utilizando topocruzamentos de soja tipo alimento x tipo grão, o objetivo do presente trabalho é: a. Determinar a herança da cor do tegumento, da cor do hilo e da expressão do caráter derramamento de hilo em populações F2:3; b. Determinação do estudo da herança da resistência ao nematoide de cisto da soja (NCS), bem como estimativa do número e modo de ação dos genes envolvidos a fim de contribuir no delineamento de cruzamentos, que envolvem os genitores em estudo, com maior possibilidade de acumular genes que conferem resistência à raça 3 do NCS e; c. Verificar com uma abordagem quantitativa, a adequação das populações a um modelo aditivo-dominante, assim como estimar parâmetros genéticos nos cruzamentos gerados. Utilizaram-se genitores contrastantes, um resistente ao NCS raça 3 ('BRSMG 810C') e dois suscetíveis, porém aptos para utilização na alimentação humana ('BRSMG 790A' e 'BRSMG 800A'). Obtiveram-se as gerações F1, RCr, RCs e F2 que foram utilizadas para o estudo da herança da resistência, por meio da segregação do caráter. As avaliações de nematoide foram realizadas utilizando-se como estimador o Índice de Fêmeas (IF) e analisadas pelo teste de Qui-Quadrado (χ2) para testar o ajuste ao modelo proposto para a herança. As sementes F3 provenientes de plantas F2 foram caracterizadas para elucidar a herança para caracteres morfológicos de interesse (cor de tegumento, cor e aspecto de hilo). Para os caracteres morfológicos dos... / Abstract: Soybean [Glycine max (L.) Merrill] is an annual leguminous that has distinct purposes, among them, a growing amount is related to the human feed. There is a demand of cultivars aiming this destination, which are resistant to pathogens and plagues. A special plague is highlighted, the soybean nematode of cyst (Heterodera glycines) (NCS). It causes expressive loss of productivity. Facing this, using soybean topcrosses feed-like x grain-like this work aims: a. determine the tegument color, hilum color, and hilum spread character heritance in F2 populations; b. determine the NCS resistance heritage, as well the involved genes number and action mode estimative in order to contribute in the crosses design, which involve the parents studied, with a higher possibility of accumulation of genes which provide resistance to the NCS race 3; and c. Verify in a quantitative approach the adequacy population in an additive-dominant model, as well estimate the genetic parameter into the crossings. We used contrasting parents, being resistant to the race 3 ('BRSMG 810C') and two susceptible, however suitable to the human feed ('BRSMG 790A' and 'BRSMG 800A'). Thus, the generations F1, RCr, RCs, and F2 were created. They were used to study heritage resistance through the character segregation. The nematode evaluations were done using as estimator the Female Index (IF) and they were analyzed by the chi-square test (χ2) to test the adjustment to the suggested model to the heritage. The F3 seeds from the F2 plants were characterized to elucidate the heritage to interest morphologic traits (seed coat color, hilum color and hilum aspect). For the morphological character of grain, the results observed of descendent were adjusted to the expected proportion, considering that there were four genes (R/I/W/T) controlling the expression coat and hilum color to both crossings. At crossing ... / Doutor

Made available in DSpace on 2014-07-29T16:24:22Z (GMT). No. of bitstreams: 1 DISSERTACAO FERNANDO G DE ARAUJO.pdf: 269926 bytes, checksum: e1a81ca1614ac2d8e004f83f9641d998 (MD5) Previous issue date: 2009-11-17 / Soybean cyst nematode, Heterodera glycines Ichinohe, is one of the major phytopathological problems affecting the soybean, in the major producing countries of this legume. Eradication of H. glycines from contaminated areas is practically impossible. Thus, the soybean growers have to adapt control measures in order to maintain low population levels. The main control measures are crop rotation, soil management and use of resistant cultivars. With the purpose to evaluate the development of H. glycines and male/female ratio on soybean resistant and susceptible cultivars two trials were conducted during the years 2007 and 2008. Susceptible cultivars BRS Aracu, BRSGO Jatai, BRSGO Luziania, BRS Favorita RR, BRS Valiosa RR, BRS Silvania RR and resistant cultivars BRSGO Ipameri e BRSGO Chapadoes were evaluated. Soybean seedlings were transplanted to pots containing naturally infested soil with H. glycines, race 14, and, 10 days later, part of the plants remained in the pots to evaluate the females, while the other part was removed for hydroponic growth to evaluate the males. In the trial of 2008 the plants were divided in three lots, with the last one having the roots stained to count the juveniles and to evaluate survival rate. Resistant cultivars always maintained a small number of females and males, except for cultivar BRSGO Ipameri that had high countings of males. Only cultivars BRS Favorita RR and BRS Silvania RR had the expected sex ratio 1:1. All other susceptible cultivars had greater number of males than females, with ratios varying from 5:1 to 11:1. Survival rate was nil for both resistant cultivars, and varied from 6.75% to 35.00% for the susceptible cultivars. Regarding the number of cysts in 100 cc of soil the only cultivar that differed significantly from the others was the BRSGO Jatai on experiment conducted in 2007. The number of eggs per cyst varied, in both experiments, and the resistance did not influence the averages found / nematóide de cisto da soja, Heterodera glycines, é um dos principais problemas fitossanitários que afeta a cultura da soja nos principais países produtores desta oleaginosa. A erradicação do H. glycines nas áreas contaminadas é praticamente impossível. Dessa forma, o produtor deve adotar práticas culturais com o intuito de manter baixos os níveis populacionais. As principais medidas de controle são a rotação de culturas, o manejo de solo e a utilização de cultivares resistentes. Com o objetivo de avaliar o desenvolvimento de H. glycines e a proporção macho:fêmea em cultivares de soja resistentes e suscetíveis foram realizados dois experimentos, sendo um em 2007 e outro em 2008. Avaliou-se as cultivares suscetíveis BRSGO Araçu, BRSGO Jataí, BRSGO Luziânia, BRS Favorita RR, BRS Valiosa RR, BRS Silvânia RR e as cultivares resistentes BRSGO Ipameri e BRSGO Chapadões. Plântulas pré-germinadas de soja foram transferidas para vasos com solo naturalmente infestado com H. glycines, raça 14, e, após dez dias, parte das plantas permaneceram nos vasos para avaliação de fêmeas e cistos no solo, e outra parte foi utilizada para instalação do sistema hidropônico para avaliação de machos. No ensaio conduzido em 2008, as plantas foram divididas em três partes sendo a última destinada à coloração de raízes para contagem de juvenis e avaliação da taxa de sobrevivência. As cultivares resistentes sempre mantiveram baixo número de fêmeas e machos com exceção da cultivar BRSGO Ipameri que obteve altos índices de machos. Somente as cultivares BRS Favorita RR e BRS Silvânia RR produziram uma relação macho/fêmea de aproximadamente 1:1. As demais cultivares suscetíveis apresentaram número de machos maior que o de fêmeas com proporções variando de 5:1 a 11:1. A taxa de sobrevivência foi nula para ambas as cultivares resistentes e variou de 6,75% a 35,00% para as cultivares suscetíveis. Com relação ao número de cistos em 100 cm³ de solo, a única cultivar que diferiu significativamente das demais foi a BRSGO Jataí no experimento realizado em 2007. O número de ovos por cisto variou bastante, em ambos os experimentos, sendo que a resistência não influenciou as médias encontradas

Soybean [Glycine max (L.) Merr.] is an important crop worldwide. It has been widely consumed for protein, oil and other soy products. To develop soybean cultivars with greater resistance against pests and improved meal quality, it is important to elucidate the molecular bases of these traits. This dissertation aims to investigate the biochemical and biological functions of soybean genes from four gene families, which are hypothesized to be associated with soybean defense against pests and soybean meal quality. There are three specific objectives in this dissertation. The first one is to determine the function of components in the salicylic acid (SA) signaling pathway in soybean resistance against soybean cyst nematode (Heterodera glycines, SCN). The second one is to determine whether insect herbivory induce the emission of volatiles from soybean, and if so, how these volatiles are biosynthesized. The third objective is to identify and characterize soybean mannanase genes that can be used for the improvement of soybean meal quality. The soybean genome has been fully sequenced, which provides opportunities for cross-species comparison of gene families of interest and identification of candidate genes in soybean. The cloned cDNAs of putative genes were expressed in Escherichia coli to produce recombinant enzymes. Through biochemical assays, these proteins were proved to be soybean salicylic acid methyltransferase (GmSAMT1), methyl salicylate esterase (GmSABP2-1), α[alpha]-farnesene synthase (GmTPS1) and E-β[beta]-caryophyllene synthase (GmTPS2), and endo-β[beta]-mannanase (GmMAN1). Through a transgenic hairy root system harboring overexpression of GmSAMT1 and GmSABP2-1, both of these two genes were evaluated for their biological function related to resistance against SCN. The results showed that the over-expression of GmSAMT1 and GmSABP2-1 in the susceptible soybean background lead to enhanced resistance against SCN. Among four putative soybean mannanase genes, one gene was cloned and characterized. GmMAN1 showed the endo-β[beta]-mannanase hydrolyse activity and can hydrolyze cell walls isolated from soybean seeds. In summary, using comparative and functional genomics, a number of genes involved in soybean defense and meal quality were isolated and characterized. This study provides novel knowledge and molecular tools for the genetic improvement of soybean for enhanced resistance and improved meal quality.

L’étude des modifications phénotypiques et génomiques associées à l’adaptation des pathogènes aux résistances est une étape fondamentale pour mieux comprendre et anticiper le phénomène de contournement des résistances. Le nématode à kyste Globodera pallida est un important pathogène de la pomme de terre, vis-à-vis duquel un QTL majeur de résistance, GpaVvrn, a été identifié chez Solanum vernei. Cependant, la capacité des populations de G. pallida à s’adapter à cette résistance en quelques générations seulement a été mise en évidence par évolution expérimentale. Dans ce contexte, ce travail de thèse avait pour objectifs (1) d’étudier les traits d’histoire de vie du nématode impactés par l’adaptation, afin de tester l’existence éventuelle d’un coût de virulence, et (2) d’identifier les régions génomiques impliquées dans l’adaptation, par une approche originale combinant évolution expérimentale et scans génomiques sur des lignées virulentes et avirulentes. Contre toute attente, nous avons montré que l’adaptation à la résistance issue de S. vernei entraînait une augmentation de la fitness des individus virulents sur hôte sensible. Nous avons également pu identifier des régions génomiques candidates à l’adaptation à la résistance de la plante hôte, contenant des gènes codant pour des effecteurs, et notamment des SPRYSECs, connus chez les nématodes à kyste pour être impliqués dans la suppression des défenses des plantes mais aussi dans la virulence du nématode. À terme, ces résultats s’avéreront utiles pour la conception de stratégies durables de déploiement de variétés de pommes de terre résistantes. / Studying phenotypic and genomic modifications associated with pathogen adaptation to resistance is a crucial step to better understand and anticipate resistance breakdown. The cyst nematode Globodera pallida is an important pest of potato crops, for which a major resistance QTL, GpaVvrn, has been identified in Solanum vernei. However, the capability of G. pallida populations to adapt to this resistance in only few generations has been highlighted through experimental evolution. In this context, the purposes of this work were (1) to study the nematode life-history traits impacted by adaptation, in order to test for potential existence of a virulence cost, and (2) to identify genomic regions involved in adaptation, through an original approach combining experimental evolution and genome scans on virulent and avirulent lineages. Unexpectedly, we highlighted that adaptation to resistance from S. vernei leads to an increase of virulent individual’s fitness on susceptible host. We were also able to pinpoint candidate genomic regions to adaptation to host plant resistance, containing genes encoding effectors, and especially SPRYSECs, known in cyst nematodes to be involved in suppression of host defense but also in nematode virulence. These results will ultimately be useful in order to conceive sustainable strategies of use of potato resistant cultivars